An electrochemical immunosensor based on ionic liquid (IL) functionalized multi-walled carbon nanotubes (MWCNTs) was developed for sensitive detection of cancer antigen 125 (CA-125) lung cancer biomarker. The IL-MWCNT nanocomposite was prepared by free radical polymerization and characterized by electron microscopy and spectroscopy. The immunosensor was fabricated by immobilizing anti-CA-125 antibodies onto IL-MWCNT modified glassy carbon electrode. Cyclic voltammetry and differential pulse voltammetry techniques were applied to optimize experimental conditions and evaluate analytical performance. The immunosensor exhibited a wide linear detection range (0.1–500 U/mL), low limit of detection (0.033 U/mL) and high reproducibility and selectivity. Testing in spiked serum samples demonstrated the sensor's potential for clinical applications. The novel IL-MWCNT nanocomposite provides an ideal interface for CA-125 capture while enhancing electrochemical properties. This electrochemical immunoassay offers a simple, rapid and ultrasensitive approach for early diagnosis of non-small cell lung cancer.